Serum response factor mediates NGF-dependent target innervation by embryonic DRG sensory neurons.

Serum response factor (SRF) is a prototypic transcription factor that mediates stimulus-dependent gene expression. Here, we show that SRF mediates NGF signaling, axonal growth, branching, and target innervation by embryonic DRG sensory neurons. Conditional deletion of the murine SRF gene in DRGs results in no deficits in neuronal viability or differentiation but causes defects in extension and arborization of peripheral axonal projections in the target field in vivo, similar to the target innervation defects observed in mice lacking NGF. Moreover, SRF is both necessary and sufficient for NGF-dependent axonal outgrowth in vitro, and NGF regulates SRF-dependent gene expression and axonal outgrowth through activation of both MEK/ERK and MAL signaling pathways. These findings show that SRF is a major effector of both MEK/ERK and MAL signaling by NGF and that SRF is a key mediator of NGF-dependent target innervation by embryonic sensory neurons.

Calcium signals control Wnt-dependent dendrite growth.

Activity-dependent dendritic growth is dependent upon intracellular calcium signaling. Yet the specific mechanisms by which calcium signals lead to morphologic changes in dendrites are not well understood. A paper in this issue of Neuron by Wayman et al. describes a novel calcium-dependent signaling cascade linking neuronal activity and calcium influx to expression of Wnt-2, a member of a family of proteins that controls elaboration of dendrites.

Cross-species annotation of basic leucine zipper factor interactions: Insight into the evolution of closed interaction networks.

Dimeric basic leucine zipper (bZIP) factors constitute one of the most important classes of enhancer-type transcription factors. In vertebrates, bZIP factors are involved in many cellular processes, including cell survival, learning and memory, cancer progression, lipid metabolism, and a variety of developmental processes. These factors have the ability to homodimerize and heterodimerize in a specific and predictable manner, resulting in hundreds of dimers with unique effects on transcription. In recent years, several studies have described dimerization preferences for bZIP factors from different species, including Homo sapiens, Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae. Here, these findings are summarized as novel, graphical representations of closed, interacting protein networks. These representations combine phylogenetic information, DNA-binding properties, and dimerization preference. Beyond summarizing bZIP dimerization preferences within selected species, we have included annotation for a solitary bZIP factor found in the primitive eukaryote, Giardia lamblia, a possible evolutionary precursor to the complex networks of bZIP factors encoded by other genomes. Finally, we discuss the fundamental similarities and differences between dimerization networks within the context of bZIP factor evolution.

A nitric oxide signaling pathway controls CREB-mediated gene expression in neurons.

Prevailing views of neurotrophin action hold that the transcription factor CREB is constitutively bound to target genes with transcriptional activation occurring via CREB phosphorylation. However, we report that within several CRE-containing genes, CREB is not constitutively bound. Upon exposure of neurons to brain-derived neurotrophic factor (BDNF), CREB becomes rapidly bound to DNA coincident with phosphorylation at its transcriptional regulatory site, Ser133. This inducible CREB-DNA binding is independent of CREB Ser133 phosphorylation and is not affected by inhibition of the ERK or PI3K signaling pathways. Instead, BDNF regulates CREB binding by initiating a nitric oxide-dependent signaling pathway that leads to S-nitrosylation of nuclear proteins that associate with CREB target genes. Pharmacological manipulation of neurons in vitro and analysis of mice lacking neuronal nitric oxide synthase (nNOS) suggest that NO mediates BDNF and activity-dependent expression of CREB target genes. Thus, in conjunction with CREB phosphorylation, the NO pathway controls CREB-DNA binding and CRE-mediated gene expression.

Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.

Alternative splicing of the P/Q-type channel (Ca(V)2.1) promises customization of the computational repertoire of neurons. Here we report that concerted splicing of its main alpha1A subunit, at both an EF-hand-like domain and the channel C terminus, controls the form of Ca2+-dependent facilitation (CDF), an activity-dependent enhancement of channel opening that is triggered by calmodulin. In recombinant channels, such alternative splicing switches CDF among three modes: (1) completely "ON" and driven by local Ca2+ influx through individual channels, (2) completely "OFF," and (3) partially OFF but inducible by elevated global Ca2+ influx. Conversion from modes 1 to 3 represents an unprecedented dimension of control. The physiological function of these variants is likely important, because we find that the distribution of EF-hand splice variants is strikingly heterogeneous in the human brain, varying both across regions and during development.

Systematic identification of splice variants in human P/Q-type channel alpha1(2.1) subunits: implications for current density and Ca2+-dependent inactivation.

P/Q-type (Ca(v)2.1) calcium channels support a host of Ca2+-driven neuronal functions in the mammalian brain. Alternative splicing of the main alpha1A (alpha1(2.1)) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human alpha1(2.1) gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca2+-dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.

Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells.

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca(2+) channel beta subunits (beta(1)-beta(4)) in cultured adult rat heart cells, to explore new dimensions of beta subunit functions in vivo. Distinct beta-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All beta-GFP subunits increased the native cardiac whole-cell L-type Ca(2+) channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between beta subunits, with a rank order of potency beta(2a) approximately equal to beta(4) > beta(1b) > beta(3). For each beta subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Q(max)) moved with depolarization. However, beta subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (P(o)). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel beta subunit, we cloned a new rat beta(2) splice variant, beta(2b), from heart using 5' rapid amplification of cDNA ends (RACE) PCR. By contrast with beta(2a), expression of beta(2b) in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into beta subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the beta subunit component of cardiac L-type Ca(2+) channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.